The epithelial cell adhesion molecule (EpCAM) is a cell surface glycoprotein, which is highly expressed on carcinoma cells. Because EpCAM is involved in cell adhesion, proliferation, survival, stemness, and tumorigenesis, it is thought to be a promising target for cancer diagnosis and therapy. Herein, we developed anti-EpCAM monoclonal antibodies (mAbs) using the Cell-Based Immunization and Screening (CBIS) method. One of the established anti-EpCAM mAb, EpMab-37 (mouse IgG₁, kappa), reacted with EpCAM-overexpressed Chinese hamster ovary-K1 cells (CHO/EpCAM) or a colorectal carcinoma cell line (Caco-2) in flow cytometry. In contrast, EpMab-37 did not react with EpCAM-knocked out Caco-2 (BINDS-16) cells in both flow cytometry and Western blot analysis. EpMab-37 could stain formalin-fixed paraffin-embedded colorectal carcinoma tissues by immunohistochemistry. Furthermore, we converted the subclass of EpMab-37 from mouse IgG₁ into IgG_2a (named as EpMab-37-mG_2a), and further produced a defucosylated version (EpMab-37-mG_2a-f), using FUT8-deficient ExpiCHO-S (BINDS-09) cells. The EpMab-37-mG_2a-f administration significantly suppressed the development of Caco-2 xenograft tumors in mice compared with the control IgG. In contrast, EpMab-37-mG_2a-f did not suppress the development of BINDS-16 xenograft tumors. These results indicated that EpMab-37 is useful for detecting EpCAM in tumors, and EpMab-37-mG_2a-f could contribute to the antibody therapy for EpCAM-positive tumors.